云南省昆明地区2021年柯萨奇病毒A10型VP1区基因特征分析

Phylogenetical analysis of coxsackievirus A10 VP1 region gene in Kunming,Yunnan province,2021

目的对云南省昆明地区2021年手足口病(hand,foot and mouth disease,HFMD)患儿粪便样本中提取的柯萨奇病毒A10型(coxsackievirus A10,CVA10)VP1区基因特征进行分析。方法从2021年昆明地区各县市区送检的手足口病患儿粪便标本中直接提取病毒RNA,先用肠道病毒EV U/EV-A71/CVA16三重核酸检测试剂盒和CVA6/CVA10病毒双通道核酸检测试剂盒进行核酸检测,检出的CVA10病毒再用486/488和487/489引物分两段扩增CVA10 VP1区全序并测序,用Sequencher 4.8软件对序列进行拼接和编辑,得到CVA10 VP1区全序列。将测得的序列与文献中的序列进行比对,用Mega 5.2软件构建系统进化树,进行基因特征及分子流行病学分析。结果2021年从852份手足口病患儿粪便标本中共检测到331株肠道病毒(enterovirus,EV),总阳性检出率为38.85%(331/852),其中,23株(6.95%,23/331)为CVA10。VP1区基因分析结果表明,23株CVA10病毒株均为基因型E,是昆明地区特有的基因型,为国内首次报道。CVA10病毒VP1区共有894个碱基,编码298个氨基酸。与原型株Kowalik进行氨基酸变异分析,结果表明,298个氨基酸位点中,昆明株分别有30~32个位点发生变化。结论云南省昆明地区2021年手足口病病原中的CVA10毒株均为基因型E,是中国新发现的基因型,应进一步加强对CVA10的基因检测和分子流行病学分析。

Objective To analyze the phylogenetic characteristics of VP1 gene of coxsackievirus group A type 10(CVA10)isolated from stool samples of hand,foot and mouth disease(HFMD)patients in Kunming,Yunnan province,China in 2021.Methods Virus RNA was directly extracted from HFMD patients stool samples.They were firstly detected by enterovirus EV U/EV-A71/CVA16 triple nucleic acid assay kit and CVA6/CVA10 virus dual channel nucleic acid assay kit.Then the detected CVA10 viruses were amplified and sequenced in two segments using primers 486/488 and 487/489,and the sequences were spliced and edited using Sequencher 4.8 software to obtain the full sequence of the CVA10 VP1 region.The VP1 sequences were compared with those in the literature and the phylogenetic tree was constructed by Mega 5.2 software for gene characterization and molecular epidemiological characteristics analysis.Results In 2021,a total of 331 strains of enterovirus(EV)were detected from 852 HFMD stool samples,with a total positive rate of 38.85%(331/852),of which,23 strains(6.95%,23/331)were CVA10.Phylogenetic analysis of the VP1 gene showed that 23 strains belonged to genotype E,a genotype unique to the Kunming area,which was first reported in China.The VP1 region of CVA10 had a total of 894 bases encoding 298 amino acids.Amino acid variation analysis with the prototype strain Kowalik showed that 30 to 32 sites were changed among 298 amino acid sites in the Kunming strain.Conclusion Kunming strains of CVA10 belonged to genotype E in 2021,which was the newer genotype in China.Strengthening the genetic testing and molecular epidemiologic surveillance is very important in the future.